Enzymes from Rhodococcus rhodochrous strain ATCC No. 53968, Bacillus sphaericus strain ATCC No. 53969 and mixtures thereof for cleavage of organic C--S bonds of carbonaceous material

ABSTRACT

An extract of membrane fragments, an enzyme, and a composition of enzymes associated with cell membranes of Rhodococcus rhodochrous strain ATCC No. 53968 and Bacillus sphaericus strain ATCC No. 53969 which have the ability to selectively react with organic sulfur of sulfur-containing organic carbonaceous material by cleavage or organic C-S bonds.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to selective removal of organically bound sulfurfrom carbonaceous materials while maintaining the calorific value of thecarbonaceous materials. The process of this invention uses bacterialproduced extracts and enzymes of Rhodococcus rhodochrous ATCC No. 53968and Bacillus sphaericus ATCC No. 53969 in removal of organic sulfur fromfossil fuels such as sulfur-containing coal and oils.

2. Description of the Prior Art

Sulfur content of carbonaceous fuels, such as coals and oils, hasprevented utilization of a considerable amount of such materials due todeleterious effect upon the environment. Inorganic pyritic sulfur andorganically bound sulfur may each constitute as much as about 3.5 weightpercent of the coal. Pyritic sulfur has been found to be relatively easyto remove by techniques including heavy media separation, selectiveagglomeration, flotation, jigging, magnetic separation, leaching andhydrosulfurization. Microbial metabolism of inorganic pyritic sulfur byits oxidation using bacteria such as Thiobacillus and Sulfolobus speciesis known. Eligwe, C. A., "Microbial Desulfurization of Coal," Fuel,67:451-458 (1988). These chemolithotropic organisms can utilizeinorganic pyritic sulfur compounds as energy sources and are capable ofremoving 90% or more of the inorganic pyritic sulfur from coal within afew days. Thiobacillus ferrooxidans is taught by U.S. Pat. No. 4,206,288as suitable for removal of pyritic sulfur from coal.

Bacillus sulfasportare ATCC 39909 has been taught by U.S. Pat. No.4,632,906 to be capable of sulfur removal from coal, withoutdifferentiation between pyritic and organic sulfur. An unidentifiedmixed culture of seven gram negative rods (ATCC 39327) prepared bygrowth in situ enriched with sulfur compounds and subsequently grown inthe presence of coal has been shown to reduce the sulfur content of coalby about 20 percent per day with a substantial portion being reductionof organic sulfur as taught by U.S. Pat. No. 4,659,670.

The feasibility of theoretical concepts of enzymatic removal of organicsulfur from coal has been suggested by Ngaiza, O. G., Wise, D. L., andGilbert, I. R., "Enzymatic Removal of Organic Sulfur from Coal," Amer.Chem. Soc. Div. Fuel Chem., 33(4) pages 623-630, (1988).

Removal of sulfur from petroleum hydrocarbons by contact with hydrogenin the presence of hydrogenase-producing microorganisms Desulfovibriodesulfuricans and Sporovibrio followed by removal of sulfur in the formof gaseous products is taught by U.S. Pat. No. 2,641,564. Removal ofsulfur from petroleum by Pseudomonas is taught by Hartdegen, F. J.,Coburn, J. M., and Roberts, R. L., "Microbial Desulfurization ofPetroleum," Chem. Eng. Progress, Vol. 80, No. 5, pp. 63-67 (1984) to beby C--C cleavage. General teachings of various Pseudomonas for removalof sulfur from petroleum are in Eckart, V., Hieke, W., Bauch., J., andGentzsch, H., "Microbial Desulfurization of Petroleum and HeavyPetroleum Fractions. 1. Studies on Microbial Aerobic Desulfurization ofRomashkino Crude Oil," Chemical Abstracts, Vol. 94, No. 142230q, (1981);Eckart, V., Hieke, W., Bauch, J., and Gentzsch, H., "MicrobialDesulfurization of Petroleum and Heavy Petroleum Fractions. 3. Change inthe Chemical Composition of Fuel-D-Oil by Microbial AerobicDesulfurization," Chemical Abstracts, Vol. 97, No. 147259c, (1982); Lee,Min Jai and Oh, Myung Soo, "Isolation, Identification, and PhysiologicalCharacteristics of Some Sulfur-Reducing Microbes," Chemical Abstracts,Vol. 78, No. 94605m (1973); Bauch, J., Gentzsch, H., Hieke, W., Eckart,V., Koehler, M., and Babenzin, H. D., "Oxidative MicrobiologicalDesulfurization of Heavy Petroleum Fractions," Chemical Abstracts, Vol.83, No. 82530y (1975); and Yuda, Sadayuki, "Petroleum Desulfurization byPseudomonas haconensis," Chemical Abstracts, Vol. 84, No. 46982j (1976).Thiobacillus thiooxidans has been identified as the most effectiveS-oxidizer and Pseudomonas putrefaciens and Desulfovibrio desulfuricansthe most effective S-reducers in microbial removal of sulfur frompetroleum, Lee, M. J., Hah, Y. C., and Lee, K. W., "Desulfurization ofPetroleum by Microorganisms. I. Isolation and Identification ofSulfur-Oxidizing and -Reducing Bacteria," Chemical Abstracts, Vol. 85,No. 156414d (1976); Lee, M. J., Hah, Y. C., and Lee, K. W.,"Desulfurization of Petroleum by Microorganisms. III. Desulfurization ofPetroleum by Contact Reaction with Desulfurizing Bacteria," ChemicalAbstracts, Vol. 85, No. 145448s (1976).

Organic sulfur which is chemically bound within the carbonaceousmolecule must be removed either by chemical or biological means.Dibenzothiophene (DBT) is the organosulfur compound most personsconsider representative of the form in which organic sulfur exists innaturally occurring organic carbonaceous fuels such as coal and oil andis the compound upon which the microbial metabolism of organosulfurcompounds has focused. Study of DBT metabolism has been pursued byseveral researchers who have isolated organisms capable of metabolizingDBT including Acinetobacter, Malik, K. A., "Microbial Removal of OrganicSulfur from Crude Oil and the Environment: Some New Perspectives,"Process Biochem., 13(9), 10-13 (1978); Arthrobacter, Knecht, A. T., Jr.,Thesis Dissertation, Louisiana State University, Order No. 621235(1961); Beijerinckia, Laborde, A. L., and Gibson, D. T., "Metabolism ofDibenzothiophene by a Beijerinckia Species," Appl. Environ. Microbiol.,34, 783-790 (1977); Rhizobium, Malik, K. A., (supra); Pseudomonas. Hou,C. T. and Laskin, A. I., "Microbial Conversion of Dibenzothiophene,"Dev. Ind. Microbiol., 17, 351-362 (1976); Isbister, J. D. andKobylinski, E. A., "Microbial Desulfurization of Coal in Processing andUtilization of High Sulfur Coals," Coal Science and Technology Series,No. 9, 627, Attia, Y. A., Ed. Amsterdam: Elsevier (1985); Knecht, A. T.,Jr., (supra); Kodama, K., Nakatani, S., Umehara, K., Shimizu, K.,Minoda, Y., and Yamada, K., "Microbial Conversion of PetrosulfurCompounds: Isolation and Identification of Products fromDibenzothiophene," Agr. Biolog. Chem., 34, 1320-1324 (1970); Monticello,D. J., Bakker, D., and Finnerty, W. R., "Plasmid Mediated Degradation ofDibenzothiophene by Pseudomonas Species," Appl. Environ. Microbiol., 49,756-760 (1985); Sulfolobus, Kargi, F. and Robinson, J. M., "MicrobialOxidation of Dibenzothiophene by the Thermophilic Organisms Sulfolobusacidocaldarius," Biotech. and Bioeng., 126, 687-690 (1984). The pathwayof microbial degradation of DBT in each of the above cases except inIsbister, et al., (supra), is by C--C bond cleavage according tomicrobial degradation pathways of DBT originally established by Kodama,et al., (supra). Microbial degradation of organic sulfur-containingcarbonaceous materials by C--C bond cleavage results in the loss of alarge portion of the calorific value of the carbonaceous fuel. Accordingto the Kodama, et al. (supra), C--C bond cleavage microbial degradationof DBT, sulfur-containing end products are 3-hydroxybenzothiophenesulfoxide, 2-formyl benzothiophene, or benzothiophene. It is, therefore,desirable to follow a microbial degradation route which removes sulfurfrom the molecule without removing carbon from the molecule, therebyretaining calorific value of the fuel to a greater degree than ispossible by carbon degradative pathways. Such sulfur-specific metabolismof the organic substrates requires cleavage of carbon-sulfur bonds inthe organic sulfur-containing molecule. In the case of sulfur specificmetabolism of dibenzothiophene, the end products are 2-hydroxybiphenyl,sulfate, and biomass. This C--S cleavage pathway is believed to proceedaccording to dibenzothiophene →dibenzothiophenesulfoxide→dibenzothiophene sulfone→dibenzothiophenesulfonate→2-hydroxybiphenyl+inorganic sulfate. The monohydroxy productof this C--S cleavage route distinguishes it from routes leading tosignificant amounts of dihydroxybiphenyl.

The only prior microorganism known to the present inventor capable ofdegradation of DBT by C--S cleavage is a Pseudomonas species asdescribed by Isbister, (supra), and Pseudomonas ATCC 39381, as set forthin U.S. Pat. No. 4,562,156. The ATCC 39381 culture on deposit does notpossess the C--S cleavage trait and the depositors of the culture havestated that the culture on deposit cannot be replaced as such cultureshaving the C--S cleavage trait to their knowledge do not exist. (4thDepartment of Energy Preparation, Utilization and Environmental ControlContractors Conference, U.S. Dept. of Energy, Pittsburgh EnergyTechnology Center, Pittsburgh, Pa. 15236, U.S.A., 1988). Mixed culturesobtained through growth under sulfur limited conditions have beencapable of selective removal of sulfur from DBT, Kilbane, John J.,"Sulfur-Specific Microbial Metabolism of Organic Compounds,"Bioprocessing of Coals Workshop, Tysons Corner, Va., Aug. 16-18, 1988.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a microbial extract,particularly those comprising membrane fragments, and a process forremoval of organically bound sulfur from sulfur-containing organiccarbonaceous materials.

It is an object of this invention to provide a microorganism membraneassociated enzyme or enzymes and a process for removal of organicallybound sulfur from sulfur-containing organic carbonaceous materials.

It is another object of this invention to provide a microbial extract,particularly those comprising membrane fragments, and process forselective sulfur removal from organic sulfur-containing fossil andfossil derived fuels.

It is another object of this invention to provide a microorganismmembrane associated enzyme or enzymes and process for selective sulfurremoval from organic sulfur-containing fossil and fossil derived fuels.

It is yet another object of this invention to provide a microbialextract, particularly those comprising membrane fragments, and processcapable of specific cleavage of C--S bonds in reactions of organiccarbonaceous materials, such as in organic synthesis and in recyclingoperations, such as recycling of rubber products.

It is yet another object of this invention to provide a microorganismmembrane associated enzyme or enzymes and process capable of specificcleavage of C--S bonds in reactions of organic carbonaceous materials,such as in organic synthesis and in recycling operations, such asrecycling of rubber products.

It is still another object of this invention to provide a microbialextract, particularly those comprising membrane fragments, and/ormembrane associated enzyme or enzymes capable of cleavage of organicC--S bonding and is stable and retains its sulfur specificcharacteristics under non-aqueous and broadened process conditions.

It is another object of this invention to provide a microbial extract,particularly those comprising membrane fragments, and/or membraneassociated enzyme or enzymes and process for specific sulfur removalfrom dibenzothiophene resulting in substantially sole products ofinorganic sulfate and 2-hydroxybiphenyl under non-aqueous and broadenedprocess conditions.

The above and other objects and advantages, as will become evident fromreading of this description, may be achieved by an extract comprisingmembrane fragments and/or membrane associated enzyme or enzymes frommutant microorganisms which have been produced and identified asRhodococcus rhodochrous ATCC No. 53968 as more fully described inallowed U.S. patent application Ser. No. 07/461,389, the disclosure ofwhich is incorporated herein by reference and Bacillus sphaericus ATCCNo. 53969 as more fully described in U.S. Pat. No. 5,002,888, thedisclosure of which is incorporated herein by reference. Each of strainsATCC No. 53968 and ATCC No. 53969 were deposited under the BudapestTreaty on the Internation Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure on Nov. 28, 1989 with American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.

Rhodococcus rhodochrous ATCC No. 53968 or Bacillus sphaericus ATCC No.53969 may be prepared by inoculating a growth medium with mixed bacteriaderived from sites having present materials of C--S bonding desired tobe cleaved, the growth medium comprising mineral nutrients, anassimilable source of carbon, and in substantial absence of asulfur-containing compound, except compounds having sulfur present onlyin C--S bonding of the type desired to be cleaved; growing the bacterialculture in the presence of oxygen at temperatures about 20° to about 34°C. and in the substantial absence of a sulfur-containing compound exceptcompounds having sulfur present only in C--S bonding of the type desiredto be cleaved for sufficient time to selectively produce Rhodococcusrhodochrous ATCC No. 53968 and/or Bacillus sphaericus ATCC No. 53969which has the property of sulfur metabolism by selective cleavage ofC--S bonds in organic carbonaceous materials. To produce Bacillussphaericus ATCC No. 53969 a helper culture may be necessary to furnishnutrients necessary for Bacillus sphaericus ATCC No. 53969 growth. Thehelper culture provides nutrient requirements for the Bacillussphaericus ATCC No. 53969, but has no ability to metabolize organicsulfur.

Extracts comprising membrane fragments may be prepared as a lysate fromthe above microorganisms by lysis processes. Any process providing aconcentration of cell membrane fragments is suitable as long aschemicals responsible for the selective cleavage of C--S bonds areretained in the product. An enzyme or enzymes associated with membranesof the above microorganisms may be separated by enzyme extractionprocesses and are capable of cleavage of C--S bonds in organiccarbonaceous materials. The enzyme or enzymes may be used in extractedform or may be further purified and used in purified form. As usedthroughout the description and claims, the terminology "enzyme" or"enzymes" is meant to include a single enzyme or a composition ofenzymes in an extracted form or purified form. Use of these sulfurspecific reactant agents of an extract comprising membrane fragmentsand/or an enzyme or enzymes associated with membranes of the specifiedmicroorganisms permits use of selective organic sulfur removal processesusing aqueous or non-aqueous media and temperatures in excess of thosewhich allow microbial growth.

According to the present invention, a sulfur specific reactant agentselected from an extract comprising membrane fragments and/or an enzymeor enzymes associated with membranes of the specified microorganisms maybe mixed directly with organic sulfur containing organic carbonaceousliquids, such as oils, or may be mixed with organic liquids forcontacting organic sulfur containing organic carbonaceous solids. Use ofthe sulfur specific reactant agent in an organic liquid avoids water/oilboundary barriers which exist when microorganism growth in an aqueousmedia is relied upon for organic sulfur removal. The sulfur specificreactant agent may be used in an aqueous medium if desired. Use ofnon-aqueous liquids is preferred since they may achieve higher catalyticrates, an expanded range of substrate utilization, and increasedstability as compared to use of aqueous media. Higher organic sulfurremoval rates may be achieved by operation of the sulfur removal processof this invention at temperatures higher than permitted whenmicroorganism growth is relied upon for sulfur removal. Additionally,organic and inorganic sulfur may be liberated and removed in a single orcontinuous non-aqueous media process.

Sulfur content of sulfur-containing organic carbonaceous material may bereduced by contacting such sulfur containing organic carbonaceousmaterial with a sulfur specific reactant agent derived from orassociated with membranes of the microorganisms Rhodococcus rhodochrousATCC No. 53968 or Bacillus sphaericus ATCC No. 53969. The process isespecially suitable for use where the sulfur-containing carbonaceousmaterial is coal or hydrocarbon oil. Such processes can result in theremoval of more than 80 percent, and preferably more than 90 percent, ofthe organically bound sulfur. The process for reducing the sulfurcontent of the sulfur-containing organic carbonaceous material occurs bycleavage of organic C--S bonds by a sulfur specific reactant derivedfrom or associated with membranes of the microorganism Rhodococcusrhodochrous ATCC No. 53968 or Bacillus sphaericus ATCC No. 53969. Thesesulfur specific reactants have the ability to selectively reduce thesulfur content of sulfur-containing organic carbonaceous material bycleavage of organic C--S bonds resulting in the production of inorganicsulfate. Extracts comprising membrane fragments and/or enzyme or enzymesassociated with membranes of derivative microorganisms of Rhodococcusrhodochrous ATCC No. 53968 or Bacillus sphaericus ATCC 53969 also havethe ability to selectively reduce the sulfur content ofsulfur-containing organic carbonaceous material by cleavage of organicC--S bonds in the same fashion as described above and are considered tobe included when the terminology "sulfur specific reactant" is used inthis description and claims.

When it is desired to selectively reduce the sulfur content of organiccarbonaceous materials having very small pore sizes, such as coal, it ispreferred to use the sulfur specific reactant enzymes and membranefragments since the smaller size of enzymes or membrane fragments ascompared with intact bacterial cells can access pores not available tothe bacteria and provide more effective contact of active materials,having the effect of increasing accessible surface area.

DESCRIPTION OF PREFERRED EMBODIMENTS

Environmental cultures having a known history of exposure toorganosulfur compounds as well as enrichment cultures using as carbonsources acetate, benzene, benzoic acid, ethanol, glucose, glycerol,nutrient broth, succinate, and toluene and organic sulfur compoundsbenzothiophene, dibenzothiophene, thiophene, trithiane, producedbacterial cultures capable of metabolizing each of the organic sulfurcompounds used. All of the environmental isolates and enrichmentcultures tested were found to metabolize organosulfur compounds byinitiating biodegradation at the carbon-carbon bond except for a mixedculture enriched with thiophene as its sole source of sulfur which wasshown to be capable of carbon-sulfur bond cleavage for about 20% of itsproducts, the remaining 80% being the result of carbon-carbon bondcleavage. The most successful microorganism for sulfur utilization fromorganosulfur compounds was Pseudomonas isolated from enrichment culturesemploying DBT as the sole source of sulfur. This Pseudomonas specieswhile capable of utilizing organically bound sulfur failed to showspecificity for the oxidation of carbon-sulfur bonds. This shows thefailure of enrichment culture development of a naturally occurringmicroorganism showing specificity for oxidation of organic C--S bonds.Thus, an unnatural, selective mutation process must be utilized todevelop a microorganism having such selective sulfur metabolism.

Microorganisms having sulfur-specific metabolic abilities with respectto organic substrates were developed by selection through a continuousculture coal bioreactor/selectostat in which nutrients and organicallybound sulfur not normally found in living tissue may be supplied in thesubstantial absence of other available sulfur such as sulfates,vitamins, amino acids and the like. The growth media should supplyorganic and inorganic nutrients for good microorganism growth, but bedevoid of inorganic and organic sulfur-containing compounds except thoseorganic sulfur-containing compounds desired to be metabolized by themutant microorganism. A suitable media for growth of microorganismsunder organosulfur conditions may suitably be composition of mineralnutrients, such as 4 gms K₂ HPO₄, 4 gms Na₂ HPO₄, 2 gms NH₄ Cl, 0.2 gmMgCl₂ ·6H₂ O, 0.001 gm CaCl₂ ·2H₂ O, and 0.001 gm FeCl₃ ·6H₂ O per literof distilled, deionized water. Any assimilable carbon source devoid ofsulfur may be used in amounts to support desired microbial growth.Suitable assimilable carbon sources include glucose, glycerol, sodiumacetate, sodium benzoate, sodium succinate, and sucrose atconcentrations of about 20 mM and benzene, ethanol, isobutanol, andtoluene may be used as vapors in the head space of the bacterial growthbioreactors. Organosulfur compounds having organic C--S bonds aresuitable, including benzothiophene, benzyldisulfide, dibenzothiophene,dibenzothiophene sulfone, phenyldisulfide, thianthrene, thioxanthene(Aldrich Chemical Company, Milwaukee, Wis.), dibenzothiophene sulfoxide(ICN Biomedicals, K&K Labs, Plainview, N.J.) and trithiane (FairfieldChemical Company, P.O. Box 20, Clythewood, S.C.) may be used overconcentration ranges which support microbial growth, in the order ofabout 20 mM and thiophene (Aldrich Chemical Company) may be used as avapor. Nutrient broth (Difco Laboratories, Detroit, Mich.) or the abovegrowth media solidified with about 15 g of agar (Difco) per liter may beemployed for streaking or plating bacterial cultures. Bacterial growthmay be monitored turbidimetrically using a Klett-Sommerson colorimeteror by enumerating colony forming units on appropriate agar.

Inoculum may be prepared by adding 5 gm samples of soil obtained fromcoal storage sites and from petroleum refinery sites to 10 ml of theabove growth media, vortexed for 60 seconds, and allowed to settle for30 minutes. The supernatants may be removed with a Pasteur pipette andused directly or diluted with an equal volume of nutrient broth andincubated at room temperature for about 24 to 48 hours before being usedto inoculate the bioreactors.

Bioreactors/selectostats were of special design to provide continuousflow of liquid nutrients while retaining coal or organosulfur solids.The same batch of coal or organosulfur compound remains within thebioreactor for the duration of its operation whereas the aqueous phasemedia may be continuously supplied to the bioreactor. The retention ofcoal within the bioreactor for long periods of time may be accomplishedby using relatively large particles of coal, typically -9+12 mesh, andthe use of an inclined, non-mixed sedimentation tube containing severalweirs/baffles from which the bioreactor effluent may be withdrawn atrelatively slow flow rates. The effluent withdrawal rates may beadjusted according to the ability of the microorganism to respond to thesulfur limitation challenge, typically, hydraulic retention times may bein the order of 72 hours.

The selectostats may be monitored frequently to determine suitablecarbon source feed rate and to assay for presence of biologicallyavailable sulfur in the effluent. This may be achieved by centrifugingfresh bioreactor effluent to remove coal fines and particles oforganosulfur substrate and bacteria followed by use of the supernatantin bacterial growth tests. Four cultures are prepared: the supernatant;the supernatant with 15 mM SO₄ ; a supernatant with 20 mM carbon source;and a supernatant with 15 mM SO₄ and 20 mM carbon source, eachinoculated with a microbial culture at 10⁵ microorganism/ml andincubated for 2 to 5 days with shaking at growth temperatures for themicroorganism being tested. Bacterial growth is monitoredturbidimetrically or by determining colony-forming units. The carbonsource sample serves to indicate the presence of biologically availablesulfur in the effluent supernatant while the sample with added sulfateserves to indicate the presence of a carbon source in the effluentsupernatant, and the sample containing both the carbon and added sulfateserves to indicate the presence of inhibitory substances in the effluentsupernatant.

The ability of bacteria to utilize organic sulfur compounds for growthcan be measured by the Sulfur Bioavailability Assay. This assay is basedon the fact that all life requires some sulfur for growth and,therefore, a situation can be created whereby quantifying bacterialgrowth provides a measure of the utilization of any organic or inorganiccompound as a source of sulfur. In practice, growth media containing acarbon source at 20 mM is used unamended, amended with 20 mM Na₂ SO₄,and amended with 20 mM of an organosulfur compound or an inorganicsulfur compound. Each of the three conditions are then inoculated with amicrobial culture at 10⁵ microorganisms/mL and incubated for 2 to 5 dayswith shaking at temperatures appropriate for the microorganism beingtested. Bacterial growth is monitored turbidimetrically or bydetermining colony forming units. The unamended sample serves as anegative control while the sample amended with sulfate serves as apositive control, and both controls are used to assess whether bacterialgrowth occurred at the expense of sulfur obtained from the organosulfurtest compound.

Development of the sulfur-specific culture may be accelerated bymutagenesis by exposure to 1-methyl-3-nitro-1-nitrosoguanidine (NTG) orto ultraviolet irradiation. Mutagenesis with NTG may be performed byspreading a solution of bacteria on an agar plate and placing a crystalof NTG in the center of the plate. During incubation, the NTG crystaldissolves in the agar forming a diffusional concentration gradient whichresults in no bacterial growth at the center and healthy growth at theouter perimeter of the plate. Between these extremes, a narrow zone ofintermediate growth is readily observable and mutagenized bacteria areobtained from this zone. Bacteria for UV-mutagenesis may be pelletedfrom liquid culture by centrifugation, washed with the above growthmedia, and resuspended in a volume of the above growth media. Threemilliliter portions may be placed in uncovered sterile petri dishes andexposed to doses of UV irradiation sufficient to cause 2 logs ofkilling, typically 10 J/m².

A mixed bacterial culture obtained from the selectostats after severalmonths operation was shown to be capable of utilizing a range oforganosulfur compounds as the sole source of sulfur as determined by theSulfur Bioavailability Assay described above. Specific C--S bondcleavage in dibenzothiophene by this mixed culture was demonstrated bygas chromatographic/mass spectrometric analysis. Standardmicrobiological techniques were used to obtain pure culturesrepresentative of each bacterial type present in the mixed culture. Eachpure culture was individually tested for its ability to utilizeorganosulfur compounds as the sole source of sulfur by the SulfurBioavailability Assay. Isolated cultures which exhibited the ability toutilize organosulfur compounds as the sole source of sulfur have beenidentified as Rhodococcus rhodochrous and Bacillus sphaericus. TheRhodococcus rhodochrous strain has been deposited with American TypeCulture Collection and assigned number ATCC 53968. The strain ischaracterized by gram positive short rods of about 0.5 μ length,producing peach-colored colonies on nutrient agar and having highorganic sulfur specificity by cleavage

of C--S bonding. The Bacillus sphaericus strain has been deposited withAmerican Type Culture Collection and assigned number ATCC 53969. Thestrain is characterized by gram positive short rods of about 0.5 μlength, producing beige/white-colored colonies on nutrient agar andhaving high organic sulfur specificity by cleavage of C--S bonding.

Bacillus sphaericus ATCC No. 53969 does not grow in chemically definedmineral salts medium in the presence of assimilable carbon and anorganosulfur compound having sulfur present only in C--S bonding withoutthe presence of a nutritional helper culture providing cross-feedingnecessary for growth. Any bacteria providing nutrients for growth undersuch conditions are satisfactory. Suitable nutritional helper culturesproviding completion of elements of nutrition for growth of Bacillussphaericus ATCC No. 53969 may be readily ascertained by one skilled inthe art. Presently known suitable helper cultures include severalEnterobacter species, such as E. aerogenes, E. agglomerans, and E.cloacae, and a Klebsiella species. The helper culture has no ability tospecifically desulfurize organic sulfur compounds.

To confirm the species identity, membrane lipids of the Rhodococcusrhodochrous ATCC 53968 were solvent extracted, derivatized and analyzedby gas chromatography. The chromatogram was compared with lipid analysesof known Rhodococcus cultures recorded in a computer library supplied byMicrocheck, Inc. (Northfield, Vt.). These tests identify ATCC 53968 asRhodococcus rhodochrous as shown by Table 1 showing all fatty acidsfound in the extract compared with the library entry listed in elutionorder in the left column. An "x" is printed for each acid on the lineopposite the fatty acid name indicating the amount of that acid and thelibrary entry mean value for the acid identified with a "+". In caseswhere the library mean percentage and the actual percentage in theextract are the same an "*" is printed. A dashed line gives a +2 or -2standard deviation window around the mean value for the library entry.Examination of Table 1 shows high certainty in the identification of theRhodococcus rhodochrous.

                                      TABLE 1                                     __________________________________________________________________________    Membrane Lipid Analysis of                                                    Rhodococcus rhodochrous ATCC No. 53968                                                     Percentage                                                       Lipid Type   05101520253035404550                                             __________________________________________________________________________    14:0 15:0 16:1 B 16:1 CIS 9 16:0 17:1 ISO 6 17:1 B unknown 16.918 17:0        18:1 ISO F 18:1 CIS 9 18:0 10 Me 18:0 19:0 20:0 SUMMED FEATURE 4 SUMMED       FEATURE 8                                                                                   ##STR1##                                                        __________________________________________________________________________

To confirm the species identity, membrane lipids of the Bacillussphaericus ATCC 53969 were solvent extracted, derivatized and analyzedby gas chromatography in the same manner. The chromatogram was comparedwith lipid analyses of known Bacillus cultures recorded in a computerlibrary supplied by Microcheck, Inc. (Northfield, Vt.). These testsidentify ATCC 53969 as Bacillus sphaericus as shown by Table 2.Examination of Table 2 shows high certainty in the identification of theBacillus sphaericus.

                                      TABLE 2                                     __________________________________________________________________________    Membrane Lipid Analysis of                                                    Bacillus sphaericus ATCC No. 53969                                                    Percentage                                                            Lipid Type                                                                            05101520253035404550556065                                            __________________________________________________________________________    14:0 ISO 14:0 15:0 ISO 15:0 ANIEISO 15:0 16:1 ISO E 16:0 ISO 16:1 A 16:0      17:1 ISO E 17:0 ISO 17:0 ANIEISO SUMMED FEATURE 5                                      ##STR2##                                                             __________________________________________________________________________

Rhodococcus ATCC 53968 was compared with other Rhodococcus speciesobtained from American Type Culture Collection with respect to carbonsources which would support the growth of these cultures. The cultureswere streaked onto the specified agar plates containing the indicatedcarbon sources and/or inoculated into liquid medium and evaluated afterincubating the cultures for 96 hours at 30° C. The results of carbonsource utilization studies using a variety of Rhodococcus strains areshown in Table 3. It appears entirely consistent from carbon sourceswhich support the growth of the microorganism that Rhodococcusrhodochrous ATCC 53968 is in fact Rhodococcus rhodochrous.

Each of the Rhodococcus species listed in Table 3 were evaluated usingthe above described sulfur bioavailability assay to determine theirability to utilize organically bound sulfur in DBT. Most strains weretested several times using a variety of substrates. The ATCC 53968strain was the only Rhodococcus species tested having the C--S bondcleavage property. Additional tests with Rhodococcus rhodochrous ATCC53968 have shown that trithiane, thianthrene, dibenzothiophene sulfoxideand dibenzothiophene sulfone and other organosulfur compounds may alsobe used as sulfur sources for Rhodococcus rhodochrous ATCC 53968.

The Rhodococcus rhodochrous ATCC 53968 strain has shown a doubling timein defined mineral salts medium in the presence of eitherdibenzothiophene or inorganic sulfate to be about 12 hours. The growthrate of Rhodococcus rhodochrous ATCC 53968 is much faster on rich medium(Luria Broth), however, other microorganisms exhibit even faster growthrates on either the defined or the rich medium.

Bacillus sphaericus ATCC 53969 was compared in the same manner asdescribed above with other Bacillus species obtained from American TypeCulture Collection with respect to carbon sources which would supportthe growth of these cultures. The results of carbon source utilizationstudies using a variety of Bacillus strains are shown in Table 4. Carbonsource utilization data obtained with Bacillus sphaericus ATCC 53969 isidentical to that obtained with Bacillus sphaericus ATCC 14577. However,a chemically defined growth medium has not been found in which Bacillussphaericus ATCC 53969 will grow as a pure culture. Additionalmicrobiological tests, including growth on nutrient agar, microscopicobservation and growth temperature studies yield identical results withboth B. sphaericus 14577 and B. sphaericus 53969. These data, inconjunction with membrane lipid analysis data, indicate that themicroorganism ATCC 53969 is a Bacillus sphaericus microorganism.

                                      TABLE 3                                     __________________________________________________________________________    Rhodococcus strains                                                                     ATCC #                                                                              Glycerol                                                                           Sucrose                                                                            Citrate                                                                            Benzoate                                                                           Acetate                                                                            Glucose                                                                            Ethanol                                                                            Succinate                                                                           Isobutanol           __________________________________________________________________________    R. rhodochrous                                                                          53968 ++   +/-  -    ++   +    +    +    -     +                    R. rhodochrous                                                                          13808 +/-  +/-  -    ++   +    +    +    +     +++                  R. rhodochrous                                                                          19149 -    +    +    ++   +    +++  +++  +     +++                  R. rhodochrous                                                                          19067 -    +    -    ++   +    +    +++  +     +++                  R. erythropolis                                                                         19369 +/-  -    +/-  -    +    ++   +++  +     +++                  R. erythropolis                                                                          4277 -    -    -    +/-  +    +    ++   +     ++                   R. globerulus                                                                           19370 +    +    +/-  ++   +    +    ++   +     ++                   R. globerulus                                                                           15903 +++  +    -    ++   +    ++   +++  +     +++                  R. equi   14887 -    +/-  -    +/-  -    +    -    -     -                    __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Bacillus strains                                                                      ATCC #                                                                             Glycerol                                                                           Sucrose                                                                            Citrate                                                                           Benzoate                                                                           Acetate                                                                            Glucose                                                                            Ethanol                                                                            Succinate                                                                          Isobutanol                __________________________________________________________________________    B. subtilis                                                                           33608                                                                              -    -    -   -    -    +    -    -    -                         B. sphaericus                                                                         14577                                                                              -    -    -   -    -    -    -    -    -                         B. sphaericus                                                                         53969                                                                              -    -    -   -    -    -    -    -    -                         __________________________________________________________________________

The Bacillus species listed in Table 4 were evaluated using the abovedescribed sulfur bioavailability assay to determine their ability toutilize organically bound sulfur in DBT. Most strains were testedseveral times using a variety of substrates. The ATCC 53969 strain wasthe only Bacillus species tested having the C--S bond cleavage property.Bacillus sphaericus ATCC No. 53969 was grown in the presence ofEnterobacter aerogenes or Enterobacter agglomerans as a nutrient helperculture. This desulfurization trait in Bacillus sphaericus ATCC 53969has been observed to be stable throughout numerous subculturing eventson both selective and non-selective medium.

The Bacillus sphaericus ATCC 53969 when grown with a helper culture inchemically defined mineral salts medium, with DBT serving as the solesource of sulfur, results in approximately 0.2 mM 2-hydroxybiphenyldetected in the medium. 2-hydroxybiphenyl is the only metabolite of DBTthat has been detected under these conditions.

The stability of the desulfurization trait of Rhodococcus rhodochrousATCC 53968 has been evaluated by growing the culture under non-selectiveconditions for multiple generations and more than 200 single colonieswere obtained and tested by the above described sulfur bioavailabilityassay, all cultures proving to be competent for desulfurization by C--Sbond cleavage. It appears that under the above culture conditions, theC--S bond cleavage ability possessed by the Rhodococcus rhodochrous ATCC53968 is a stable trait. The desulfurization trait of Rhodococcusrhodochrous ATCC 53968 is maintained through heat shocking. Growth ofRhodococcus rhodochrous ATCC 53968 is severely retarded or absent at 37°and 42° C. when incubated at those temperatures for 48 hours.Seventy-two single colonies of desulfurization competent Rhodococcusrhodochrous ATCC 53968 have been streaked onto nutrient agar, incubatedat 37° C. or 42° C. for 48 hours followed by incubation at 30° C. for 72hours and tested by the above described sulfur bioavailability assay,with all colonies exhibiting stable maintenance of the desulfurizationtrait.

The mutant culture Rhodococcus rhodochrous ATCC 53968 was inoculatedinto a mineral salts glucose bacterial growth medium containingdibenzothiophene as the sole sulfur source. After growth at 30° C. for72 hours, the cultures were centrifuged, the supernatants processed bysolid-phase extraction using C-18 silica compounds, eluted withdichloromethane, and analyzed using gas chromatography/mass spectrometryto identify and quantify the metabolites of dibenzothiophene. Theresults are shown in Table 5, quantitation of the metabolites beingaccomplished by spiking the dichloromethane eluates with a knownconcentration of ethylnapthalene and comparing metabolite peaks withretention times and concentration curves prepared with pure chemicalcompounds. Compounds that were specifically looked for are listed inTable 5. The fact that only 2-hydroxybiphenyl was found and that3-hydroxy-2-formyl-benzothiophene and any other compounds known to beformed in the carbon destructive pathway of DBT degradation were notfound demonstrates that the Rhodococcus rhodochrous ATCC 53968metabolizes dibenzothiophene via C--S bond cleavage route and not by aC--C bond cleavage route as do many of the microorganisms of the priorart.

                  TABLE 5                                                         ______________________________________                                        METABOLITES OF RHODOCOCCUS                                                    RHODOCHROUS ATCC 53968                                                        Compound               Mol. Wt. ppm                                           ______________________________________                                         *Dibenzothiophene-5-oxide                                                                           200      BDL                                             plus Phenoxathiin    200                                                    **Dihydroxybiphenyl    186      BDL                                           **2-hydroxybiphenyl    170      35.27                                         + 3-hydroxy-2-formyl-benzothiophene                                                                  178      BDL                                           **Biphenyl             154      BDL                                           + Benzothiophene       134      BDL                                           + Three isomers of C.sub.8 H.sub.6 OS:                                         (hydroxybenzothiophene)                                                       No. 1                 150      BDL                                            No. 2                 150      BDL                                            No. 3                 150      BDL                                           + C.sub.9 H.sub.8 OS   164      BDL                                           + C.sub.9 H.sub.8 O.sub.2 S                                                                          180      BDL                                           + C.sub.9 H.sub.6 OS   162      BDL                                           + C.sub.10 H.sub.10 OS or C.sub.9 H.sub.6 O.sub.2 S                                                  178      BDL                                           +C.sub.8 H.sub.8 O.sub.2 S isomers                                             a)                    168      BDL                                            b)                    168      BDL                                           + Formula (?)          220      BDL                                           ______________________________________                                         *C-S cleavage intermediate                                                    **C-S cleavage product                                                        + C-C cleavage product                                                        BDL means below detection level of ˜0.001                          

Rhodococcus rhodochrous ATCC 53968 derivatives have been shown to retainthe same or better selective desulfurization trait of the Rhodococcusrhodochrous ATCC 53968 strain. An antibiotic resistant derivative ofRhodococcus rhodochrous ATCC 53968 was used in mixed culture withEnterobacter aerogenes. The mixed inoculum contained a tenfold excess ofRhodococcus rhodochrous ATCC 53968 relative to E. aerogenes and growthwas monitored in the above defined growth medium, glycerol, and DBT asthe sole source of sulfur. At 50, 70, 140 and 240 hours samples of theculture were used to prepare dilution series which were plated ontonutrient agar and nutrient agar containing 250 micrograms/mLstreptomycin. E. aerogenes grew faster than the Rhodococcus rhodochrousATCC 53968 derivative on the nutrient agar while only the Rhodococcusrhodochrous ATCC 53968 derivative grew on nutrient agar containingantibiotic. The Rhodococcus rhodochrous ATCC 53968 derivative grewrapidly during the first 70-80 hours since it is the only organismcapable of metabolizing sulfur from DBT. However, after about 50-60hours, even though E. aerogenes cannot metabolize DBT, it begins rapidgrowth and at 240 hours is present in nearly three orders of magnitudegreater abundance than the Rhodococcus rhodochrous ATCC 53968derivative. This suggests that DBT is metabolized in association withthe outer cell membrane of the Rhodococcus rhodochrous ATCC 53968bacteria in a manner such that sulfur liberated from DBT by Rhodococcusrhodochrous ATCC 53968 is available for metabolism by other bacteria.

Bacillus sphaericus ATCC 53969 derivatives retain the same or betterselective desulfurization trait of the ATCC 53969 strain. Bacillussphaericus ATCC 53969 derivatives having the same selectivedesulfurization trait are suitable for use in any of the describeddesulfurization processes and are intended to be included for such uses.

The desulfurization trait of Bacillus sphaericus ATCC 53969 isapparently associated with the outer cell membrane of thismicroorganism. This fact is supported by the observation that the helperculture, as a pure culture, has no ability to grow in chemically definedmineral salts medium in which DBT serves as the sole sulfur source.However, when a desulfurization competent microorganism, specificallyBacillus sphaericus ATCC 53969, is simultaneously present, this helperculture grows profusely. This profuse growth of the helper culture couldonly occur if sulfur liberated from DBT by Bacillus sphaericus ATCC53969 was made available for use by the helper culture.

The sulfur content of sulfur-containing organic carbonaceous materialsmay be selectively reduced by contacting with a sulfur specific reactantagent. The sulfur specific reactant agent according to this inventionmay be an extract comprising membrane fragments and/or an enzyme orcomposition of enzymes associated with a cell membrane wherein the cellmembrane is from microorganisms Rhodococcus rhodochrous strain ATCC No.53968 and/or Bacillus sphaericus strain ATCC No. 53969. These sulfurspecific reactant agents may be used in aqueous or non-aqueous media forthe highly efficient removal of organic sulfur from organicsulfur-containing carbonaceous materials, particularly naturallyoccurring fossil fuels such as coal, petroleum, shale, oil, lignite, andsynthetic fuels derived therefrom.

Suitable non-aqueous media include organic liquids, such as, kerosene,crude oils, petroleum distillates, vegetable oils, and other light oils,glycerol, dimethylformamide, methanol, ethanol, benzene, toluene,octanol, octane, ethyl acetate, and hexane. Preferred organic liquidsinclude kerosene, light oils, and methanol.

Extracts comprising membrane fragments may be prepared as a lysate fromthe above microorganisms by lysis processes, such as sonication, use ofdetergents, or use of a French press, as well known in the art. Anyprocess providing a concentration of cell membrane fragments is suitableas long as chemicals responsible for the selective cleavage of C--Sbonds are available in the product.

Enzymes associated with membranes of the above microorganisms may beseparated by enzyme extraction processes and are capable of cleavage ofC--S bonds in organic carbonaceous materials. Suitable enzyme extractionprocesses include lysis by sonication, detergents or by a French pressfollowed by ammonium sulfate precipitation, fractionation, gelpermeation chromatography, electrophoresis, isoelectric focusing, highpressure liquid chromatography, liquid chromatography, affinitychromatography, immunoprecipitation, or other suitable procedures, aswell known in the art. The enzyme or enzymes may be used in extractedform or may be further purified and used in purified form.

Use of extracts comprising membrane fragments and/or enzymes associatedwith membranes of the specified microorganisms permits use of selectiveorganic sulfur removal processes using non-aqueous media andtemperatures in excess of those which allow microbial growth. Theorganic sulfur is preferably selectively removed from organicsulfur-containing carbonaceous materials by contacting with the sulfurspecific reactant agent in an organic medium for increased contactcompatibility for a time sufficient to remove a substantial portion ofthe organic sulfur. According to the present invention, the sulfurspecific reactant agent may be mixed directly with organic sulfurcontaining organic carbonaceous liquids, such as oils, or may be mixedwith organic liquids for contacting organic sulfur containing organiccarbonaceous solids. The sulfur specific reactant agent should be usedin an amount sufficient to provide a concentration effective toselectively react with a substantial portion of the organic sulfurwithin the time limitations of the process. Use of the sulfur specificreactant agent according to this invention in organic liquid mediaavoids water/oil boundary barriers which exist when microorganism growthand/or use of microorganisms or their products in an aqueous media isrelied upon for organic sulfur removal. The sulfur specific reactantagent may be used in an aqueous medium if desired. Use of non-aqueousliquids is preferred since they achieve higher catalytic rates, anexpanded range of substrate utilization, and increased stability ascompared to use of aqueous media. Higher organic sulfur removal ratesmay be achieved by operation of the sulfur removal process attemperatures higher than permitted when microorganism growth is reliedupon for sulfur removal. Sulfur removal reaction rates may be increasedat temperatures greater than about 35° C., about 35° to about 100° C.being preferred. Additionally, organic and inorganic sulfur may beremoved in a single or continuous non-aqueous media process. Any mannersof contacting to result in highly efficient chemical reaction may beused as known to the art, for example, ground coal may be agitated in anoil based liquid medium of extract comprising membrane fragments fromRhodococcus rhodochrous ATCC No. 53968 at a temperature of about 50° C.for a time sufficient to obtain conversion of more than about 80 percentof the organic sulfur to inorganic sulfate.

Sulfur content of sulfur-containing organic carbonaceous material may bereduced by contacting such sulfur containing organic carbonaceousmaterial with the sulfur specific reactant agent according to thisinvention. The process is especially suitable for use where thesulfur-containing carbonaceous material is coal or hydrocarbon oil. Suchprocesses can result in the removal of more than 80 percent, andpreferably more than 90 percent, of the organically bound sulfur.

The selective sulfur reactant agents according to this inventionuniquely react with sulfur by cleavage of the C--S bonding in organiccarbonaceous materials; for example, in reaction with dibenzothiophene,the sole products are 2-hydroxybiphenyl and inorganic sulfate. Theseproperties render the selective sulfur reactant agents of this inventionspecific agents for use in organic chemical synthesis for cleavage oforganic C--S bonding which may be used in various organic processsynthesis systems. Likewise, the selective sulfur reactant agents ofthis invention may be utilized in desulfurizing degradation of a widevariety of organic materials by cleavage of organic C--S bonding inrecycling operations, such as in breakdown of sulfur containing organicmolecules such as in rubber products.

The process of this invention results in the conversion of organicsulfur to inorganic sulfate. Sulfur in the form of organically boundsulfur presents very difficult separation, while the inorganic sulfateproduced by this process may be easily removed by a wide variety ofmethods readily apparent to one of ordinary skill in the art.

Biological membranes are generally hydrophobic and therefore extractscomprising membrane fragments and enzymes associated with membranes cannaturally prefer non-aqueous systems. Extracts comprising membranefragments and enzymes associated with membranes of the sulfur selectivemicroorganisms Rhodococcus rhodochrous strain ATCC No. 53968 andBacillus sphaericus strain ATCC No. 53969 according to the presentinvention can offer improved performance and process flexibility, suchas the potential for simultaneous removal of both organic and inorganicsulfur from carbonaceous materials. Catalytic rates can be increased asmuch as several hundredfold, the range of substrates acted upon can beexpanded, and tolerance to pH, temperature, and other environmentalstresses can be dramatically improved by use of membrane fragments andenzymes in organic liquids. For example: the activity of peroxidasecatalyzed pyrogallol oxidation is increased one hundredfold usingreversed micelles of the enzyme in octanol versus its activity in water,Martinek, K., Levashov, A. V., Klyachko, N., Khmelnitski, Y. L., andBerezin, I. V., "Micellar Enzymology", J. Biochem., 155, 453-468 (1986);the use of enzymes in non-aqueous or low-aqueous systems is reviewed inKhmelnitsky, Y. L., Levashov, A. V., Klyachko, N. L., and Martinek, K.,"Engineering Biocatalytic Systems in Organic Media with Low WaterContent," Enzyme Micro. Technol., 10 (December, 1988); and biologicalreactions in treatment of coal using reverse micelle solutions showedcell-free enzyme extracts of T. ferrooxidans cells outperformed thewhole-cell preparations, Lee, K. I. and Yen, T. F., "CoalDesulfurization Through Reverse Micelle Biocatalysis Process," ACS Div.of Fuel Chemistry, Int'l. Symposium, Los Angeles, Sep. 25-30, 1988.

The process of this invention may be advantageously used in conjunctionwith other processes to provide integrated processes, such as: removalof both inorganic and organic sulfur; and use of a chemical and/orphysical process to expand the pore structure of coal to provide greateractive surface area for more effective sulfur removal.

While in the foregoing specification this invention has been describedin relation to certain preferred embodiments thereof, and many detailshave been set forth for purpose of illustration it will be apparent tothose skilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

I claim:
 1. A composition comprising membrane fragments extracted from amicroorganism selected from the group consisting of Rhodococcusrhodochrous strain ATCC NO. 53968, Bacillus sphaericus strain ATCC No.53969, and mixtures thereof, said membrane fragments containing enzymeshaving the ability to selectively react with sulfur of sulfur-containingorganic carbonaceous material by cleavage of organic C--S bonds.
 2. Acomposition according to claim 1 wherein said microorganism isRhodococcus rhodochrous strain ATCC No.
 53968. 3. A compositionaccording to claim 1 wherein said microorganism is Bacillus sphaericusstrain ATCC No.
 53969. 4. A composition according to claim 1 whereinsaid membrane fragments are in an organic liquid medium.
 5. Acomposition according to claim 1 wherein said membrane fragments are inan organic carbonaceous liquid.
 6. A composition according to claim 1wherein said membrane fragments are in an organic carbonaceous oil.
 7. Acomposition according to claim 1 wherein said membrane fragments are inan aqueous medium.
 8. A composition according to claim 1 wherein saidcarbonaceous material is coal.
 9. A composition according to claim 1wherein said carbonaceous material is hydrocarbon oil.